Combination chemotherapy compositions and methods

ABSTRACT

This invention relates to combination therapies involving anticancer chemotherapeutic agents and isoflavones or analogues thereof. The invention flier relates to compounds, compositions, methods and therapeutic uses involving, containing, comprising, including and/or for preparing platinum-isoflavonoid complexes suitable for use in the combination therapies of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-In-Part of U.S. application Ser. No. 10/530,176, filed Mar. 9, 2006; which is a 371 of PCT/AU03/01296, filed Oct. 2, 2003; the disclosures of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to combination therapies involving anticancer chemotherapeutic agents and isoflavones or analogues thereof. The invention further relates to compounds, compositions, methods and therapeutic uses involving, containing, comprising, including and/or for preparing platinum-isoflavonoid complexes suitable for use in the combination therapies of the invention.

BACKGROUND

The regulation of cell division (mitosis) is of critical importance to the normal growth and development of a multicellular organism, as well as the homeostatic maintenance of tissues, and the ability of certain cell types to respond appropriately to environmental cues.

Oncogenesis is the process by which normal cells lose the ability to regulate progression through the cell cycle via “checkpoints” (mitosis), and the ability to undergo apoptosis due to acquired or inherited damage to regulatory genes. Those cells containing aberrant genetic information may undergo clonal expansion and act as a site for additional genetic alteration, at which time uncontrolled proliferation results in the formation of a neoplasm.

Neoplasms are generally classified as benign or malignant. Benign tumours proliferate locally and are composed of differentiated cells resembling those of the tissue of origin, the edge of the tumour remaining well defined, and usually encapsulated. Malignant neoplasms (classically termed “cancers”) are not encapsulated and their edges are ill-defined, the cells are also less well differentiated than the cells of origin, and show increased mitotic activity.

Localised, chronic irritation or inflammation can also cause cells to divide abnormally, resulting in abnormal growths or cellular masses, or tumours. Reactive cellular growth responses to clearly defined, chronic irritant stimulation are described as metaplasias. In dysplasias, there is a disorganisation of the pattern of squamous epithelium in tissues such as the skin, oesophagus and uterus in response to chronic irritation or inflammation.

Numerous compounds are commercially available as chemotherapeutic agents for destruction of abnormally proliferating cells in benign and malignant neoplasias, dyplasias and metaplasias. Predicting the responsiveness of a given tumour-related disease type to a particular drug is difficult, as each disease type is different and may respond to different treatments. Generally, clinical treatment of cancer and other cellular proliferative disorders involves having different chemotherapy treatment options for each condition.

An example of an important chemotherapeutic agent is the platinum-based compound cisplatin (cis-diamminedichloroplatinum (H); cis-Cl₂(NH₃)Pt). Cisplatin has a square planar geometry, with each of the two chloride groups (and likewise, each of the two amine groups) being adjacent, or cis, to each other.

Cisplatin was first approved for human use in the late 1970's and is prescribed for the treatment of a variety of tumours including germ-cell, advanced bladder carcinoma, adrenal cortex carcinoma, breast, testicular and ovarian cancer, head and neck carcinoma and lung carcinoma.

Cisplatin is active against proliferating or cancerous cells by binding to DNA and interfering with its repair mechanism, eventually leading to cell death. It is thought that the first step in the cellular process is that a molecule of water replaces one of the chloride ions of cisplatin. The resulting intermediate structure can then bind to a single nitrogen on a DNA nucleotide. Following that, the second chloride is also replaced by another water molecule and the platinum agent then binds to a second nucleotide. Binding studies of cisplatin with DNA have indicated a preference for nitrogen 7 on two adjacent guanines on the same strand. It also binds to adenine and across strands to a lesser extent.

The binding of cisplatin to DNA causes production of intrastrand cross-links and formation of DNA adducts. The adducts or cisplatin-DNA complexes attract the attention of DNA repair proteins which become irreversibly bound, The resulting distortion to the shape of the DNA by the binding of cisplatin prevents effective repair and hence, cell death.

Other well known chemotherapeutic agents include carboplatin, the taxanes such as paclitaxel, docetaxel, gemcitabine, 5-fluorouracil, methotrexate and the tetracyclines.

Patients undergoing cancer chemotherapy often have to contend with quite severe and debilitating side effects due to the toxicity of the active agents. Common side effects of chemotherapy are nausea and vomiting. Other side effects include temporary reduction in bone marrow function, numbness or tingling in hands or feet, changes in hearing, temporary taste alterations, loss of appetite, diarrhoea and allergic reactions.

Chemotherapy regimes are further complicated by the efficacy of currently available chemotherapeutic agents against various cancers or other tumour types sometimes being insufficient. For example, some cancer cells have developed natural tolerance against the therapeutic agents. Further, some therapeutic or prophylactic agents exert side effects, or can induce the development of tolerance in abnormally dividing cells during clinical use, leading to a situation in which certain tumour types become multiply drug resistant. Multidrug resistance thus remains a main complication of long-term successful tumour chemotherapy.

Melanoma is one of the most prevalent cancers in Australia and the incidence of melanoma is on the rise around the world. Indeed, in the United States the incidence of melanoma is rising faster than that of any other malignancy. However, melanoma is typically resistant to standard chemotherapy and radiation therapy. A number of chemotherapeutic agents, including platinum-based and taxane drugs have been used to treat melanoma but with disappointing response rates. Our understanding of the resistance mechanisms employed by melanoma to avoid the cytotoxic effects of conventional therapeutics is limited, as is our ability to devise alternative therapeutics to overcome resistance.” The increased incidence of melanoma, compounded by the lack of effective therapy, in particular once the disease has metastasized, underscores the need for improved methods of treating patients with melanoma.

Accordingly there is a strong need to identify new, improved and/or alternative pharmaceutical compositions, agents and treatment regimes against chemoresistance, mutated growth or proliferation of cells in cancer and other age-related diseases. There is a further need for chemotherapeutic agents which address undesirable side effects associated with known agents. There is also an everpresent need for physicians to have at their disposal alternative therapeutic options for the treatment of malignancies that display innate or acquired resistance to currently available therapeutics. Agents which can act synergistically with other chemotherapeutics thereby improving efficacy are highly sought after. Agents that enhance the efficacy of conventional therapeutics, in conjunction with reducing dosage and treatment regimens, and enhance the targeting of malignant cells, will hopefully result in fewer side-effects commonly associated with standard chemotherapy.

It is an object of the present invention to provide pharmaceutical compositions and methods for the treatment, amelioration or prophylaxis of cancer and diseases associated with oxidant stress. The present invention also seeks to provide pharmaceutical compositions and methods for targeting neoplastic cells for treatment, which compositions and methods provide improved cell activity in terms of targeting function, improved delivery of toxic agents and/or improvement or restoration of chemosensitivity.

SUMMARY OF THE INVENTION

This application now describes new treatment regimes and chemotherapeutic compositions and compounds. The invention is based on the totally unexpected activity of isoflavonoid compounds in restoring or, addressing the chemo-selectivity or activity of anticancer agents, synergistic compositions including same and novel isoflavonoid-drug complexes.

According to an aspect of the present invention there is provided a method of increasing the sensitivity of cancer cells or a tumour to a chemotherapeutic agent by contacting said cells or tumour with an isoflavonoid compound of formula (I) as set out below.

Compounds of the general formula (I) are the isoflavonoid compounds represented by the formula:

in which

-   -   R₁, R₂ and Z are independently hydrogen, hydroxy, OR₉, OC(O)R₁₀,         OS(O)R₁₀, CHO, C(O)R₁₀, COOH, CO₂R₁₀, CONR₃R₄, alkyl, haloalkyl,         arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl,         alkoxyaryl, thio, alkylthio, amino, alkylamino, dialkylamino,         nitro or halo, or     -   R₂ is as previously defined, and R₁ and Z taken together with         the carbon atoms to which they are attached from a five-membered         ring selected from     -   R₁ is as previously defined, and R₂ and Z taken together with         the carbon atoms to which they are attached form a five-membered         ring selected from         and     -   W is R₁, A is hydrogen, hydroxy, NR₃R₄ or thio, and B is         selected from         W is R₁, and A and B taken together with the carbon atoms to         which they are attached form a six-membered ring selected from     -   W, A and B taken together with the groups to which they are         associated are selected from     -   W and A taken together with the groups to which they are         associated are selected from         and B is selected from         wherein     -   R₃ is hydrogen, alkyl, arylalkyl, alkenyl, aryl, an amino acid,         C(O)R₁₁ where R₁₁ is hydrogen, alkyl, aryl, arylalkyl or an         amino acid, or CO₂R₁₂ where R₁₂ is hydrogen, alkyl, haloalkyl,         aryl or arylalkyl,     -   R₄ is hydrogen, alkyl or aryl, or     -   R₃ and R₄ taken together with the nitrogen to which they are         attached comprise pyrrolidinyl or piperidinyl,     -   R₅ is hydrogen, C(O)R₁₁ where R₁₁ is as previously defined, or         CO₂R₁₂ where R₁₂ is as previously defined,     -   R₆ is hydrogen, hydroxy, alkyl, aryl, amino, thio, NR₃R₄, COR₁₁         where R₁₁ is as previously defined, CO₂R₁₂ where R₁₂ is as         previously defined or CONR₃R₄,     -   R₇ is hydrogen, C(O)R₁₁ where R₁₁ is as previously defined,         alkyl, haloalkyl, alkenyl, aryl, arylalkyl or Si(R₁₃)₃ where         each R₁₃ is independently hydrogen, alkyl or aryl,     -   R₈ is hydrogen, hydroxy, alkoxy or alkyl,     -   R₉ is alkyl, haloalkyl, aryl, arylalkyl, C(O)R₁₁ where R₁₁ is as         previously defined, or Si(R₁₃)₃ where R₁₃ is as previously         defined,     -   R₁₀ is hydrogen, alkyl, haloalkyl, amino, aryl, arylalkyl, an         amino acid, alkylamino or dialkylamino,     -   the drawing         represents either a single bond or a double bond,     -   T is independently hydrogen, alkyl or aryl,     -   X is O, NR₄ or S, and     -   Y is:         wherein     -   R₁₄, R₁₅ and R₁₆ are independently hydrogen, hydroxy, OR₉,         OC(O)R₁₀, OS(O)R₁₀, CHO, C(O)R₁₀, COOH, CO₂R₁₀, CONR₃R₄, alkyl,         haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, thio,         alkylthio, amino, alkylamino, dialkylamino, nitro or halo, or         any two of R₁₄, R₁₅ and R₁₆ are fused together to form a cyclic         alkyl, aromatic or heteroaromatic structure, and         pharmaceutically acceptable salts thereof

In a preferred embodiment the cancer cells or tumour are pre-treated with a compound of formula (I), prior to treatment with the chemotherapeutic agent.

In another embodiment, the compound of formula (I) is administered concurrently with the. chemotherapeutic agent.

In a further embodiment the compound of formula (I) is administered after resistance to a chemotherapeutic agent is observed in cancer cells and tumours, and in particular after multidrug resistance is observed.

In a further embodiment the sensitivity of the cancer cells or tumour to the chemotherapeutic agent is restored or regenerated.

In an embodiment, the cancer cells are melanoma cells and the tumour is a melanoma.

According to another aspect there is provided a combination therapy comprising administering to a subject a therapeutically effective amount of a compound of formula (I) and a chemotherapeutic agent.

Combination therapy in accordance with the invention is typically for the treatment or prevention of cell proliferation and cancers including benign prostatic hypertrophy; melanoma; breast cancer; uterine cancer; ovarian cancer; testicular cancer; large bowel cancer; endometrial cancer; prostatic cancer; uterine cancer; and diseases associated with oxidant stress including cancer, myocardial infarction stroke, arthritis, sunlight induced skin damage or cataracts (for convenience hereafter referred to as the “therapeutic indications”).

In a preferred embodiment the administration of the compound of formula (I) precedes the administration of the chemotherapeutic agent. Alternatively, the administration is concurrent. In a further embodiment the combination therapy follows observed resistance by cancer cells and tumours to a chemotherapeutic agent or agents.

In a preferred embodiment the subject cell growth is proliferation, and the subject down-regulation is killing off the proliferating cells. The condition being treated is typically cancer, more typically a metastatic cancer. The cancer may be selected from melanoma, breast cancer, prostatic cancer, testicular cancer, ovarian cancer, uterine cancer and/or colorectal cancer, and more preferably is ovarian cancer, prostatic cancer or pancreatic cancer.

In a further aspect of the present invention there is provided a method for the treatment of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) and a chemotherapeutic agent.

According to a further aspect of the present invention there is provided a method for increasing the sensitivity of melanoma cells or a melanoma to a chemotherapeutic agent by contacting said cells or melanoma with an isoflavonoid compound of formula (I).

In an embodiment, the chemotherapeuatic agent is carboplatin. In an embodiment, the compound of formula (I) is dehydroequol.

According to a further aspect of the preset invention there is provided a method for the treatment of melanoma in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) and a chemotherapeutic agent.

In further aspects of the invention there is provided methods for the manufacture of medicaments for the above stated methods of the invention and pharmaceutical agents useful for same.

This application also describes new therapeutic compositions and complexes comprising platinum-based pharmaceutical agents. The invention is based on the totally unexpected biological activity of new platinum-isoflavonoid complexes and of isoflavonoid compounds of formula (I) which form synergistic compositions or complexes with platinum-based chemotherapeutic agents.

Accordingly, a further aspect of the invention provides a pharmaceutical composition for the treatment or prevention of cell proliferation and cancers, the composition comprising at least one isoflavonoid compound of formula (I) and at least one chemotherapeutic agent.

In an embodiment, the cancer is melanoma. In an embodiment, the chemotherapeutic agent is selected from: carboplatin, cisplatin, paclitaxel, docatexel, gemcitabine and topotecan. In an embodiment, the compound of formula (I) is dehydroequol.

The compositions and platinum-isoflavonoid complexes are important targeting agents for the delivery of toxic signals to cells. The compositions and methods of the invention are directed to treating a condition in a subject, which condition is characterised by the undesirable, detrimental or otherwise unwanted growth or proliferation of cells.

Also disclosed herein are platinum-isoflavonoid complexes and analogues thereof described by general formula (II):

in which

-   -   R_(A), R_(B), R_(C), and R_(D) are independently halo, hydroxy,         XR_(E), alkoxy, OC(O)R_(F), OS(O)R_(F), thio, alkylthio, amino,         alkylamino or dialkylamino,     -   X is O, NR_(F) or S, and     -   R_(F) is hydrogen, alkyl, arylalkyl, alkenyl, aryl or an amino         acid,         wherein

-   at least one of R_(A), R_(B), R_(C), and R_(D), and preferably only     R_(A), is XR_(E) where R_(E) is an isoflavonoid compound represented     by general formula (I) set out above or is derived from or is a     radical or ion of the isoflavonoid compound (I) and ligates to the     platinum through any one or more of the heteroatoms X or a radical     of the heteroatoms defined as part of R_(E) or alternatively by a     double bond on the isoflavonoid compound (I) and

-   when R_(A) is XR_(E), R_(B), R_(C) and/or R_(D) together may form     part of a bidentate or tridentate ligand of general formulae (B)     and (T) respectively

-   wherein L represents a ligating atom chosen from N, O and S,

-   n is from 0 to 8, and

-   each R6 is independently as defined above or may together form part     of a cyclic alkyl, aromatic or heteroaromatic structure,

-   which platinum-isoflavonoid complexes include pharmaceutically     acceptable salts thereof.

It has also surprisingly been found by the inventors that platinum-isoflavonoid complexes of the general formula (II) have particular utility and effectiveness in the treatment or prevention of the therapeutic indications noted above.

Thus also disclosed herein is a method for, the treatment or prevention of the therapeutic indications described above which method comprises administering to a subject a therapeutically effective amount of one or more platinum-isoflavonoid complexes of the formula (II) as defined above.

Also disclosed herein is a method of treating a condition in a mammal, which condition is characterised by the undesirable, detrimental or otherwise unwanted growth of cells, said method comprising administering to said mammal an effective amount a platinum-isoflavonoid complex of formula (II) for a time and under conditions sufficient to down-regulate the growth of said cells.

In a preferred embodiment the subject cell growth is proliferation, and the subject down-regulation is killing off the proliferating cells. The condition being treated is typically cancer, more typically a metastatic cancer. The cancer may be selected from melanoma, breast cancer, prostatic cancer, testicular cancer, ovarian cancer, uterine cancer and/or colorectal cancer, and more preferably is ovarian cancer, prostatic cancer or pancreatic cancer.

Also disclosed herein is a method of down-regulating the growth of cells, said method comprising contacting said cells with an effective amount of a platinum-isoflavonoid complex of formula (II).

In a preferred embodiment the subject cell growth is proliferation, and the subject down-regulation is killing off the proliferating cells.

Also disclosed herein is the use of platinum-isoflavonoid complexes of the formula (II) for the manufacture of a medicament for the treatment or prevention of one or more of the therapeutic indications.

Also disclosed herein is the use of one or more platinum-isoflavonoid complexes of the formula (II) in the treatment or prevention of one or more of the therapeutic indications.

Also disclosed herein is an agent for the treatment or prevention of the therapeutic indications which comprises one or more platinum isoflavonoid complexes of the formula (II) either alone or in association with one or more carriers or excipients.

Also disclosed herein is a therapeutic composition which comprises one or more platinum-isoflavonoid complexes of the formula (II) in association with one or more pharmaceutical carriers and/or excipients.

Also disclosed herein is a drink or food-stuff, which contains one or more platinum-isoflavonoid complexes of the formula (II).

Also disclosed herein are compositions comprising a platinum complex of the general formula (IIa),

in which

-   -   R_(G), R_(H), R_(I),and R_(J) are independently halo, hydroxy,         alkoxy, OC(O)R_(K), OS(O)R_(K), thio, alkylthio, amino,         alkylamino or dialkylamino,     -   X is O, NR_(K) or S, and     -   R_(K) is hydrogen, alkyl, arylalkyl, alkenyl, aryl or an amino         acid, or a pharmaceutically acceptable salt thereof, and an         isoflavonoid compound of general formula (I) as defined above.

These compositions comprising a platinum complex of the formula (IIa) and an isoflavonoid compound of the formula (I) are found to have particular utility, effectiveness and synergism in the treatment or prevention of the therapeutic indications set out above.

Thus also disclosed herein is a method for the treatment or prevention of the therapeutic indications which comprises administering to a subject a therapeutically effective amount of compositions comprising a platinum complex of the formula (IIa) in conjunction with an isoflavonoid compound of formula (I).

Also disclosed herein is the combined use of a platinum complex of the formula (IIa) and an isoflavonoid compound of the formula,(I) in the manufacture of a medicament for the treatment or prevention of the therapeutic indications.

Also disclosed herein is the use of a platinum complex of the formula (IIa) and an isoflavonoid compound of the formula (I) in the treatment or prevention of the therapeutic indications.

Also disclosed herein is a kit comprising a platinum complex of the formula (IIa) and an isoflavonoid compound of the formula (I) either alone or in association with one or more carriers or excipients.

Also disclosed herein is an agent for the treatment, or prevention of the therapeutic indications which comprises a composition comprising a platinum complex of the formula (IIa) and an isoflavonoid compound of the formula (I) either alone or in association with one or more carriers or excipients.

Throughout this specification and the claims which follow, unless the text requires otherwise, the word “comprise”, and variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents the cell viability of various cancer cell lines over different concentrations of carboplatin.

FIG. 2 represents the cell viability of various cancer cell lines over different concentrations of paclitaxel.

FIG. 3 represents the cell viability of various cancer cell lines over different concentrations of carboplatin following dehydroequol pre-treatment.

FIG. 4 represents the cell viability of various cancer cell lines over different concentrations of paclitaxel following dehydroequol pre-treatment.

FIG. 5 represents a Western Blot analysis of carboplatin or paclitaxel treatment resistant ovarian cancer CP70 cells with and without dehydroequol pre-treatment.

FIG. 6 represents tumour mass comparison of dehydrequol and cisplatin when delivered as single active agents or combination therapy with the 5% HPBCD vehicle control group.

FIG. 7 represents tumour volume comparison of dehydrequol and cisplatin when delivered as single active agents or combination therapy with the 5% HPBCD vehicle control group.

FIG. 8 represents a body weight comparison as an indicator of toxicity in each dehydroequol, cisplatin or combination treatment group in comparison with the HPBCD 5% vehicle control.

FIG. 9 represents unpaired t-tests showing differences in expression of XIAP between benign, primary, metastatic, and all malignant (primary and metastatic) melanoma specimens.

FIG. 10 represents a cell viability assay showing the effect of dehydroequol on three melanoma cell lines, YUGEN8, YUMAC, and YUSAC. Each experiment was performed in triplicate and viability was determined by the CellTiter 96 Aqueous Once Solution Cell Proliferation Assay. Cells were treated with 10 ug/mL of dehydroequol for 24 hours.

FIG. 11 represents the effect of dehydroequol (Pxd) on YUMAC (A)and YUSAC (B) cells. Cells were treated with a single dose of dehydroequol (10 ug/mL) for varying time points (0, 4, 8, and 24 hours), XIAP expression was determined by western blot analysis. Caspase-3,-8, and -9 activities were determined by the Caspase-Glo 3, 8, and 9 assays, respectively.

FIG. 12 represents cell viability assays demonstrating the effect of dehydroequol on melanoma cells. (A) Cells were treated with increasing doses of Carboplatin (50-200 ug/mL) alone or (B) cells were pretreated with 10 ug/mL dehydroequol for 4 hours followed by treatment with increasing doses of Carboplatin. Each experiment was performed in triplicate and viability was determined by the CellTiter 96 Aqueous One Solution Cell Proliferation Assay, reported as a percentage of viable cells relative to untreated cells.

FIG. 13 represents the effect of pretreatment with dehydroequol on the apoptotic cascade. YUMAC cells received either no treatment, 200 ug/mL of Carboplatin for 24 hours, 10 ug/mL of dehydroequol for 4 hours, or 10 ug/mL of dehydroequol for 4 hours followed by 200 ug/mL Carboplatin for 24 hours. Both pro- and antiapoptotic protein expression was determined by Western blot analysis. Activity of caspases -3,-8, and -9 were determined using the Caspase-Glo 3, 8, and 9 assays, respectively.

FIG. 14 represents the in vivo sensitizing effect of dehydroequol on A2780 mouse xenograft model. (A) Tumor mass measured in animals treated with vehicle (Group 1), dehydroequol (Pxd) 25 mg/kg (Group 2), Topotecan 2 mg/kg (Group 3), Topotecan 1 mg/kg (Group 4), Pxd 25 mg/kg +Topotecan 2 mg/kg (Group 5), or Pxd 12.5 mg/kg+Topotecan 1 mg/kg (Group 6). *p<0.01 group 5 vs 3 and group 6 vs 3;**p<0.01 group 2 vs 1 (B) Comparative mean terminal tumor mass taken from the same groups of animals. Inset shows tabulated mean tumor mass data and calculated %T/C values. *p<0.01 (group 4 vs 3); **p<0.01 group 6 vs 4);***p<0.01 group 2 vs 1. Eight animals were tested in each group.

DETAILED DESCRIPTION OF THE INVENTION

The terms “isoflavonoid”, “isoflavonoid” and “isoflavone” as used herein are to be taken broadly to include ring-fused benzopyran molecules having a pendent phenyl group from the pyran ring based on a 1,2-diphenylpropane system. Thus, the classes of compounds generally referred to as isoflavones, isoflavenes, isoflavans, isoflavanones, isoflavanols and the like are generically referred to herein as isoflavones, isoflavone derivatives or isoflavonoid compounds.

The term “alkyl” is taken to mean both straight chain and branched chain alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, teriary butyl, and the like. The alkyl group has 1 to 10 carbon atoms, preferably from 1 to 6 carbon atoms, more preferably methyl, ethyl propyl or isopropyl. The alkyl group may optionally be substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, C₁-C₄-alkoxycarbonyl, C₁-C₄-alkylamino-carbonyl, di-(C₁-C₄-alkyl)-amino-carbonyl, hydroxyl, C₁-C₄-alkoxy, formyloxy, C₁-C₄-alkyl-carbonyloxy, C₁-C₄-alkylthio, C₃-C₆-cycloalkyl or phenyl.

The term “aryl” is taken to include phenyl and naphthyl and may be optionally substituted by one or more C₁-C₄-alkyl, hydroxy, C₁-C₄-alkoxy, carbonyl, C₁-C₄-alkoxycarbonyl, C₁-C₄-alkylcarbonyloxy or halo.

The term “halo” is taken to include fluoro, chloro, bromo and iodo, preferably fluoro and chloro, more preferably fluoro. Reference to for example “haloalkyl” will include monohalogenated, dihalogenated and up to perhalogenated alkyl groups. Preferred haloalkyl groups are trifluoromethyl and pentafluoroethyl.

The term “pharmaceutically acceptable salt” refers to an organic or inorganic moiety that carries a charge and that can be administered in association with a pharmaceutical agent, for example, as a counter-cation or counter-anion in a salt. Pharmaceutically acceptable cations are known to those of skilled in the art, and include but are not limited to sodium, potassium, calcium, zinc and quaternary amine. Pharmaceutically acceptable anions are known to those of skill in the art, and include but are not limited to chloride, acetate, citrate, bicarbonate and carbonate.

The term “pharmaceutically acceptable derivative” or “prodrug” refers to a derivative of the active compound that upon administration to the recipient, is capable of providing directly or indirectly, the parent compound or metabolite, or that exhibits activity itself. Prodrugs are included within the scope of the present invention.

As used herein, the terms “treatment” and “prevention” and the like are to be considered in their broadest context. In particular, the term “treatment” does not necessarily imply that an animal is treated until total recovery. Accordingly, “treatment” includes amelioration of the symptoms or severity of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.

As used herein the term “therapeutically effective amount” includes within its meaning a non-toxic but sufficient amount of an agent(s) or compound(s) to provide the desired therapeutic or preventative effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent(s) being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact “therapeutically effective amount”. However, for any given case, an appropriate “therapeutically effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.

Preferred isoflavonoid compounds of formula (I) are selected from general formulae (III)-(IX), and more preferably are selected from general formulae (IV)-(IX):

in which

-   -   R₁, R₂, R₅, R₆, R₁₄, R₁₅, W and Z are as defined above,         more preferably     -   R₁, R₂, R₁₄, R₁₅, W and Z are independently hydrogen, hydroxy,         OR₉, OC(O)R₁₀, C(O)R₁₀, COOH, CO₂R₁₀, alkyl, haloalkyl,         arylalkyl, aryl, thio, alkylthio, amino, alkylamino,         dialkylamino, nitro or halo,     -   R₅ is hydrogen, C(O)R₁₁ where R₁₁ is hydrogen, alkyl, aryl, or         an amino acid, or CO₂R₁₂ where R₁₂ is hydrogen, alkyl or aryl,     -   R₆ is hydrogen, hydroxy, alkyl, aryl, COR₁₁ where R₁₁ is as         previously defined, or CO₂R₁₂ where R₁₂ is as previously         defined,     -   R₉ is alkyl, haloalkyl, arylalkyl, or C(O)R₁₁ where R₁₁ is as         previously defined, and     -   R₁₀ is hydrogen, alkyl, amino, aryl, an amino acid, alkylamino         or dialkylamino,         more preferably     -   R₁ and R₁₄ are independently hydroxy, OR₉, OC(O)R₁₀ or halo,     -   R₂, R₁₅, W and Z are independently hydrogen, hydroxy, OR₉,         OC(O)R₁₀, C(O)R₁₀, COOH, CO₂R₁₀, alkyl, haloalkyl, or halo,     -   R₅ is hydrogen, C(O)R₁₁ where R₁₁ is hydrogen or alkyl, or         CO₂R₁₂ where R₁₂ is hydrogen or alkyl,     -   R₆ is hydrogen or hydroxy,     -   R₉ is alkyl, arylalkyl or C(O)R₁₁ where R₁₁ is as previously         defined, and     -   R₁₀ is hydrogen or alkyl,         and more preferably     -   R₁ and R₁₄ are independently hydroxy, methoxy, benzyloxy,         acetyloxy or chloro,     -   R₂, R₁₅, W and Z are independently hydrogen, hydroxy, methoxy,         benzyloxy, acetyloxy, methyl, trifluoromethyl or chloro,     -   R₅ is hydrogen or CO₂R₁₂ where R ₁₂ is hydrogen or methyl, and     -   R₆ is hydrogen.

Particularly preferred isoflavonoid compounds of formula (I) are selected from:

In a further embodiment the preferred isoflavonoid compounds are the isoflav-3-ene and isoflavan compounds of general formula (VI), and more preferred are the 3-ene compounds of the general formula (VIa):

in which

-   -   R₁, R₂, R₆, R₁₄, R₁₅, W and Z are as defined above;         more preferably     -   R₁, R₂, R₁₄, R₁₅, W and Z are independently hydrogen, hydroxy,         OR₉, OC(O)R₁₀, C(O)R₁₀, COOH, CO₂R₁₀, alkyl, haloalkyl,         arylalkyl, aryl, thio, alkylthio, amino, alkylamino,         dialkylamino, nitro or halo,     -   R₆ is hydrogen, hydroxy, alkyl, aryl, COR₁₁ where R₁₁ is as         previously defined, or CO₂R₁₂ where R₁₂ is as previously         defined,     -   R₉ is alkyl, haloalkyl, arylalkyl, or C(O)R₁₁ where R₁₁ is as         previously defined, and     -   R₁₀ is hydrogen, alkyl, amino, aryl, an amino acid, alkylamino         or dialkylamino,         more preferably     -   R₁ is hydroxy, OR₉, OC(O)R₁₀ or halo,     -   R₂, R₁₄, R₁₅, W and Z are independently hydrogen, hydroxy, OR₉,         OC(O)R₁₀, C(O)R₁₀, COOH, CO₂R₁₀, alkyl, haloalkyl, or halo,     -   R₆ is hydrogen,     -   R₉ is alkyl, arylalkyl or C(O)R₁₁ where R₁₁ is as previously         defined, and     -   R₁₀ is hydrogen or alkyl,         and more preferably     -   R₁ is hydroxy, methoxy, benzyloxy, acetyloxy or chloro,     -   R₂, R₁₄, R₁₅, W and Z are independently hydrogen, hydroxy,         methoxy, benzyloxy, acetyloxy, methyl, trifluoromethyl or         chloro, and     -   R₆ is hydrogen,     -   including pharmaceutically acceptable salts and derivatives         thereof.

In a most preferred embodiment of the invention the isoflavonoid compound is dehydroequol, Cpd. 12. As such, particular reference is made to dehydroequol in the description, Examples which follow and accompanying drawings however this is not to be taken as being unnecessarily limiting on the disclosure of the invention provided herein.

Chemotherapeutic agents are generally grouped as DNA-interactive agents, antimetabolites, tubulin-interactive agents, hormonal agents, other agents such as asparaginase or hydroxyurea. Each of the groups of chemotherapeutic agents can be further divided by type of activity or compound. Chemotherapeutic agents used in combination with the isoflavonoid compound of formula (I) of the present invention, or salts thereof of the present invention, may be selected from any of these groups but are not limited thereto. For a detailed discussion of the chemotherapeutic agents and their method of administration, see Dorr, et al, Cancer Chemotherapy Handbook, 2d edition, pages 15-34, Appleton and Lang (Connecticut, 1994) herein incorporated by reference.

DNA-interactive agents include alkylating agents, e.g. cisplatin, cyclophosphamide, altretamine; DNA strand-breakage agents, such as bleomycin; intercalating topoisomerase I and II inhibitors, e.g., topotecan, dactinomycin and doxorubicin); nonintercalating topoisomerase I and II inhibitors such as, etoposide and teniposide; the DNA minor groove binder plicamydin, for example, and nucleoside analogs which inhibit DNA synthesis, such as gemcitabine and flurouracil.

The alkylating agents form covalent chemical adducts with cellular DNA, RNA, or protein molecules, or with smaller amino acids, glutathione, or similar chemicals. Generally, alkylating agents react with a nucleophilic atom in a cellular constituent, such as an amino, carboxyl, phosphate, or sulfhydryl group in nucleic acids, proteins, amino acids, or in glutathione. The mechanism and the role of these alkylating agents in cancer therapy is not well understood.

Typical alkylating agents include, but are not limited to, nitrogen mustards, such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, uracil mustard; aziridine such as thiotepa; methanesulphonate esters such as busulfan; nitroso ureas, such as carmustine, lomustine, streptozocin; platinum complexes, such as cisplatin, carboplatin; bioreductive alkylator, such as mitomycin, and procarbazine, dacarbazine and altretamine.

DNA strand breaking agents include bleomycin, for example.

DNA topoisomerase II inhibitors include the following intercalators, such as amsacrine, dactinomycin, daunorubicin, doxorubicin (adriamycin), idarubicin, and mitoxantrone; nonintercalators, such as etoposide and teniposide, for example.

Antimetabolites interfere with the production of nucleic acids by one of two major mechanisms. Certain drugs inhibit production of deoxyribonucleoside triphosphates that are the immediate precursors for DNA synthesis, thus inhibiting DNA replication. Certain of the compounds are analogues of purines or pyrimidines and are incorporated in anabolic nucleotide pathways. These analogues are then substituted into DNA or RNA instead of their normal counterparts.

Antimetabolites useful herein include, but are not limited to, folate antagonists such as methotrexate and trimetrexate; pyrimidine antagonists, such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine antagonists include mercaptopurine, 6-thioguanine, fludarabine, pentostatin; and ribonucleotide reductase inhibitors include hydroxyurea.

Tubulin interactive agents act by binding to specific sites on tubulin, a protein that polymerizes to form cellular microtubules. Microtubules are critical cell structure units. When the interactive agents bind the protein, the cell can not form microtubules. Tubulin interactive agents include the vinca alkaloids vincristine and vinblastine, paclitaxel (Taxol) and docetaxel, for example.

Hormonal agents are also useful in the treatment of cancers and tumors. They are used in hormonally susceptible tumors and are usually derived from natural sources. Hormonal agents include, but are not limited to, estrogens, conjugated estrogens and ethinyl estradiol and diethylstilbesterol, chlorttianisen and idenestrol; progestins such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens such as testosterone, testosterone propionate; fluoxymesterone, and methyltestosterone.

Adrenal corticosteroids are derived from natural adrenal cortisol or hydrocortisone. They are used because of their anti-inflammatory benefits as well as the ability of some to inhibit mitotic divisions and to halt DNA synthesis. These compounds include, but are not limited to, prednisone, dexamethasone, methylprednisolone, and prednisolone.

Leutinizing hormone releasing hormone agents or gonadotropin-releasing hormone antagonists are used primarily the treatment of prostate cancer. These include leuprolide acetate and goserelin acetate. They prevent the biosynthesis of steroids in the testes.

Antihormonal antigens include, for example, antiestrogenic agents such as tamoxifen, antiandrogen agents such as flutamide, and antiadrenal agents such as mitolane and aminoglutethimide.

Further agents include the following: hydroxyurea appears to act primarily through inhibition of the enzyme ribonucleotide reductase, and asparaginase is an enzyme which converts asparagine to nonfunctional aspartic acid and thus blocks protein synthesis in the tumour.

Preferred chemotherapeutic agents for use in the subject invention are cisplatin, carboplatin, taxol (paclitaxel), docataxel, fluorouracil, gemcitabine, fluxuridine, cyclophosphamide ifosfamide, hexamethylmelamine, estramustine, mitomycin, topotecan and docetaxel.

Compounds of formula (I) also e chemotherapeutic activity and in this regard particular reference can be made to dehydroequol, Cpd. 12.

Preferred bidentate and tridentate platinum ligands of the present inventions include those commonly known in the art. For example, suitable bidentate ligands may be selected from ethylene-1,2-diamine and 1,10-phenathraline and other ligands well known in the art.

Preferred platinum complexes are halo and amino substituted, more preferably chloro and amine substituted, more preferably cis-dichlorodiamino substituted. Preferred platinum-isoflavonoid complexes are preferably halo and amino substituted, more preferably cis-dichloroamino substituted or cis-diaminochloro substituted.

Compounds of the present invention have particular application in the treatment of diseases associated with or resulting from estrogenic effects, androgenic effects, vasolidatory and spasmodic effects, inflammatory effects and oxidative effects.

The amount of compounds of formulae (I), (II) or (I) and (IIa) which are required in a therapeutic treatment according to the invention will depend upon a number of factors, which include the specific application, the nature of the particular compound used, the condition being treated, the mode of administration and the condition of the patient. Compounds of formulae I or Ia and II may be administered in a manner and amount as is conventionally practised. See, for example, Goodman and Gilman, The Pharmacological Basis of Therapeutics, 1299 (7th Edition, 1985). The specific dosage utilised will depend upon the condition being treated, the state of the subject, the route of administration and other well known factors as indicated above. In general, a daily dose per patient may be in the range of 0.1 mg to 10 g; typically from 0.5 mg to 1 g; preferably from 50 mg to 200 mg. Importantly the synergistic relationship of the isoflavonoid compounds of general formula (I) and the chemotherapeutic agent allow for significant reductions in dosage regimes of relatively toxic drugs such as cisplatin, paclitaxel and carboplatin for example.

Other preferred dosage regimes and amounts are set out in the Examples and accompanying drawings.

The production of a pharmaceutical composition for the treatment of the therapeutic indications herein described (for convenience hereafter referred to as the “active compounds”) are typically admixed with one or more pharmaceutically or veterinarially acceptable carriers and/or excipients as are well known in the art.

The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. The carrier or excipient may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose, for example, a tablet, which may contain from 0.5% to 59% by weight of the active compound, or up to 100% by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well known techniques of pharmacy consisting essentially of admixing the components, optionally including one or more accessory ingredients.

The formulations of the invention include those suitable for oral, rectal, optical, buccal (for example, sublingual), parenteral (for example, subcutaneous, intramuscular, intradermal, or intravenous) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.

Formulation suitable for oral administration may be presented in discrete units, such as capsules, sachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture such as to form a unit dosage. For example, a tablet may be prepared by compressing or moulding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the compound of the free-flowing, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Moulded tablets may be made by moulding, in a suitable machine, the powdered compound moistened with au inert liquid binder.

Formulations suitable for buccal (sublingual) administration include lozenges comprising the active compound in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.

Compositions of the present invention suitable for parenteral administration conveniently comprise sterile aqueous preparations of the active compounds, which preparations are preferably isotonic with the blood of the intended recipient. These preparations are preferably administered intravenously, although administration may also be effected by means of subcutaneous, intramuscular, or intradermal injection. Such preparations may conveniently be prepared by admixing the compound with water or a glycine buffer and rendering the resulting solution sterile and isotonic with the blood. Injectable formulations according to the invention generally contain from 0.10/% to 60% w/v of active compound(s) and are administered at a rate of 0.1 ml/minute/kg or as appropriate. Parenteral administration is a preferred route of administration for the compounds of the present invention.

Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

Formulations or compositions suitable for topical administration to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include Vaseline, lanoline, polyethylene glycols, alcohols, and combination of two or more thereof. The active compound is generally present at a concentration of from 0.1% to 0.5% w/w, for example, from 0.5% to 2% w/w. Examples of such compositions include cosmetic skin creams.

Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches suitably contain the active compound as an optionally buffered aqueous solution of, for example, 0.1 M to 0.2 M concentration with respect to the said active compound.

Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6), 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable formulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water and contain from 0.1 M to 0.2 M active ingredient.

The active compounds may be provided in the form of food stuffs, such as being added to, admixed into, coated, combined or otherwise added to a food stuff, The term food stuff is used in its widest possible sense and includes liquid formulations such as drinks including dairy products and other foods, such as health bars, desserts, etc. Food formulations containing compounds of the invention can be readily prepared according to standard practices.

Therapeutic methods, uses and compositions may be for administration to humans and other animals, including mammals such as companion and domestic animals (such as dogs and cats) and livestock animals (such as cattle, sheep, pigs and goats), birds (such as chickens, turkeys, ducks) and the like.

The active compound or pharmaceutically acceptable derivatives prodrugs or salts thereof can also be co-administered with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, antiinflammatories, or antiviral compounds. The active agent can comprise two or more isoflavones or derivatives thereof in combination or synergistic mixture. The active compounds can also be administered with lipid lowering agents such as probucol and nicotinic acid; platelet aggregation inhibitors such as aspirin; antithrombotic agents such as coumadin; calcium channel blockers such as verapamil, diltiazem, and nifedipine; angiotensin converting enzyme (ACE) inhibitors such as captopril and enalapril, and β-blockers such as propanolol, terbutalol, and labetalol. The compounds can also be administered in combination with nonsteriodal antiinflammatories such as ibuprofen, indomethacin, aspirin, fenoprofen, mefenamic acid, flufenamic acid and sulindac. The compounds can also be administered with corticosteroids.

The co-administration may be simultaneous or sequential. Simultaneous administration may be effected by the compounds being in the same unit dose, or in individual and discrete unit doses administered at the same or similar time. Sequential administration may be in any order as required and typically will require an ongoing physiological effect of the first or initial active agent to be current when the second or later active agent is administered, especially where a cumulative or synergistic effect is desired.

The isoflavones of formula (I) for use in the present invention may be derived from any number of sources readily identifiable to a person skilled in the art. Preferably, they are obtained in the form of concentrates or extracts from plant sources. Again, those skilled in the art will readily be able to identify suitable plant species, however, for example, plants of particular use in the invention include leguminous plants. More preferably, the isoflavone extract is obtained from chickpea, lentils, beans, red clover or subterranean clover species and the like.

Isoflavone extracts may be prepared by any number of techniques known in the art. For example, suitable isoflavone extracts may be prepared by water/organic solvent extraction from the plant source. It will be appreciated that an isoflavone extract may be prepared from any single tissue of a single species of plant or a combination of two or more different tissues thereof. Similarly, an extract may be prepared from a starting material which contains a heterogeneous mixture of tissues from two or more different species of plant.

Generally, where an isoflavone extract is prepared from plant material, the material may be comminuted or chopped into smaller pieces, partially comminuted or chopped into smaller pieces and contacted with water and an organic solvent, such as a water miscible organic solvent. Alternatively, the plant material is contacted with water and an organic solvent without any pre-treatment. The ratio of water to organic solvent may be generally in the range of 1:10 to 10:1 and may, for example, comprise equal proportions of water and solvent, of 1% to 30% (v/v) organic solvent. Any organic solvent or a mixture of such solvents may be used. The organic solvent may preferably be a C2-10, more preferably a C1-4 organic solvent (such as methanol, chloroform, ethanol, propanol, propylene glycol, erythrite, butanol, butanediol, acetonitrile, ethylene glycol, ethyl acetate, glycidol, glycerol dihydroxyacetone or acetone). Optionally the water/organic solvent mixture may include an enzyme which cleaves isoflavone glycosides to the algycone form. The mixture may be vigorously agitated so as to form an emulsion. The temperature of the mix may range, for example, from an ambient temperature to boiling temperature. Exposure time may range from one hour to several weeks. One convenient extraction period is twenty-four hours at 90° C. The extract may be separated from undissolved plant material and the organic solvent removed, such as by distillation, rotary evaporation, or other standard procedures for solvent removal. The resultant extract containing water soluble and non-water soluble components may be dried to give an isoflavone-containing extract, which may be formulated with one or more pharmaceutically acceptable carriers, excipients and/or auxiliaries according to the invention.

An extract made according to the description provided in the previous paragraphs may contain small amounts of oil which include isoflavones in their aglycone form (referred to herein as isoflavones). This isoflavone enriched oil, may be subject to HPLC to adjust the isoflavone ratios, or, if it is at the desired isoflavone ratio, may be dried, for example in the presence of silica, and be formulated with one or more carriers, excipients and/or auxiliaries to give an isoflavone containing extract. Alternatively, the isoflavones contained in said small amounts of oil may be further concentrated by addition to the oil of a non-water soluble organic solvent such as hexane, heptane, octane acetone or a mixture of one or more of such solvents. One example is 80% hexane, 20% acetone w/w having high solubility for oils but low solubility for isoflavones. The oil readily partitions into the organic solvent, and an enriched isoflavone containing extract falls out of solution. The recovered extract may be dried, for example in an oven at 50° C. to about 120° C., and formulated with one or more pharmaceutically acceptable carriers, excipients and/or auxiliaries.

It will be appreciated that the present invention also contemplates the production of suitable isoflavones, functional derivatives, equivalents or analogues thereof, by established synthetic techniques well known in the art. See, for example, Chang et al. (1994) which discloses methods appropriate for the synthesis of various isoflavones.

International Patent Applications WO 98/08503 and WO 00/49009 (which are incorporated herein in their entirety by reference) and references cited therein also provide general synthetic methods for the preparation of isoflavonoid compounds for use in the present invention.

General methods known in the art may also be employed by those skilled in the art of chemical synthesis for constructing the platinum complexes depicted m formula (II), and by reference to the general schemes 1 and 2 below.

Chemical functional group protection, deprotection, synthons and other techniques known to those skilled in the art may be used where appropriate in the synthesis of the compounds of the present invention.

The inventors have found a surprising synergy between the compounds of formula (I), and in particular the isoflav-3-ene compounds of formula (VIa), with known chemotherapeutic agents. The isoflavonoid compounds of the invention are found to restore or at least improve chemosensitivity to previously resistant cancer cell lines. In particular, dehydroequol (12, DHE) is found to exhibit synergistic interaction with cisplatin, carboplatin, topotecan and paclitaxel with various established cancer cell lines, in particular the ovarian cancer cell lines Cp70 and A27A0. Synergism was also observed with prostate cancer cell lines DU145 and PC3 and pancreatic cell line HPAC.

These results are further elucidated in the examples which follow. These results show that combination chemotherapy with the isoflavonoid compounds with established anticancer agents are useful in the treatment of proliferation of cancer cells and neoplastic tumours by reducing the IC₅₀ of standard chemotherapy. Administration of the isoflavonoid compounds described herein either simultaneously, sequentially or as a pre-treatment to standard chemotherapies increases the sensitivity of the cancer cells and tumours to chemotoxic agents.

The Examples show the efficacy of combination chemotherapy with dehydroequol as a treatment for epithelial ovarian cancer cells by such reduction of the IC50 of standard chemotherapy. This thereby increases sensitivity of the cancer cells to chemotoxic agents. The results of these tests and trial are important as ovarian cancer is the fourth leading cause of cancer death and the most lethal of the gynaecologic malignancies. Recent new therapies have led to some improvement in the five year survival, yet there has been no improvement in the overall survival. Be main limitations of therapy in ovarian cancer patients are chemoresistance and side-effects. The combination chemotherapy and isoflavonoid pre-treatment addresses the survival rates of patients undergoing the chemotherapy, and in particular those patients with ovarian cancer. Without wishing to be limited to theory, it is believed that the isoflavone derivative dehydroequol induces apoptosis in ovarian cancer cells by specifically removing the blockers of apoptosis.

The invention is further described with reference to the following non-limiting examples.

EXAMPLE 1 Dehydroequol-Cisplatin Synergy in vitro

The effect of a composition comprising the platinum complex cisplatin and the isoflavonoid compound dehydroequol (compound No. 12) on various cancer cell lines was assessed on culture plates. Cell viability was determined using CellTiter©. Apoptosis was evaluated using Hoechst 33342 dye.

It was found that the amount of cisplatin needed to kill a set number of cancer cells is less when in admixture with an isoflavonoid compound as compared to a control with cisplatin alone. This example demonstrates the surprising synergy between cisplatin and the isoflavonoid compounds of the present invention. Dehydroequol was found to exhibit a strong synergistic interaction with cisplatin in cell lines derived from ovarian (A2780, Cp70), prostate (DU145 and PC3) and pancreatic (HPAC) cancers. Table 1 below shows that the IC₅₀ of cisplatin against the mentioned cell lines is markedly lowered by co-incubating representative cells with a sub-IC₅₀ level (2 μM) of dehydroequol. TABLE 1 Effect of concurrent exposure to dehydroequol and cisplatin on the IC₅₀ levels on nominated cancer cell lines IC₅₀ (uM) Cisplatin IC₅₀ (uM) + Cell line Cisplatin dehydroequol 2 uM dehydroequol A2780 3.0 1.7 <0.001 CP70 10.4 1.5 0.1 HPAC 34.5 50.0 7.7 PC3 0.4 9.6 0.001 DU145 5.0 5.9 0.1

EXAMPLE 2 Dehydroequol-Cisplatin, Dehydroequol-Carboplatin and Dehydroequol-Paclitaxel Synergy in vitro and in vivo

Methods

The in vitro studies were performed using ovarian cancer cells isolated from ascites using an immunomagnetic assay and established ovarian cancer cell lines CP70 and A2780. Cell viability was determined using CellTiter©. Apoptosis was evaluated using Hoechst 33342 dye. The in vivo effect was tested by injecting CP70 subcutaneously into nude mice. Animals received daily oral administration of dehydroequol, 10 or 20 mg/kg for 8 days alone or in combination with cisplatin 0.5 mg/kg. After 8 days the animals were sacrificed and the tumour volume was measured.

The IC50 for carboplatin ranged from 60 μg/ml to greater than 100 μg/ml (FIG. 1).

The IC50 for paclitaxel in the paclitaxel resistant cell line, R182, was greater than 2 μM (FIG. 2).

Pre-treatment with dehydroequol (10 μg/ml) for two hours significantly reduced the IC50 for carboplatin (0.5 μg/ml+/−0.5) and paclitaxel (0.05 μM) (FIGS. 3 and 4).

Western blot analysis demonstrated that resistant ovarian cancer cells expressed high levels of active XIAP (X-linked inhibitor of apoptosis). Additionally, the active form of caspase 3 in chemoresistant cells was not detected. Caspase 3 activation was observed in the chemoresistant cells only after pretreatment with dehydroequol (FIG. 5).

FIGS. 6 and 7 depict the results of the next study, where 20 mg/kg dehydroequol (DHE) 5% HPBCD was compared to delivery of cisplatin and to a combination of dehydroequol and cisplatin. The 20 mg/kg cisplatin dosage regimen inhibited tumour proliferation but the data was not significantly different from cisplatin (1 mg/kg) and dehydroequol (20 mg/kg) controls. Importantly and somewhat surprisingly, the lower dose 10 mg/kg dehydroequol-0.5 mg/kg cisplatin combination regimen inhibited tumour proliferation more markedly than that over the 20 mg/kg dehydroequol-1 mg/kg cisplatin (%T/C=14.7) regimen and the data were significantly different from single agent controls (FIGS. 6 and 7).

Dehydroequol treatment for 48 hours (h) induced 60-80% decrease in cell viability in carboplatin and paclitaxel resistant cells. Pre-treatment with pH alone for 2 h decreased cell viability by 20%. Furthermore, pretreatment (2 h) with pH in chemoresistant cells followed by carboplatin or paclitaxel for 48 h resulted in a 30% and 50% significant decrease in cell viability, respectively, Hoechst stain confirmed the presence of apoptosis in the treated cells. In vivo, cisplatin (0.5 mg/kg) had no effect on tumour size while the combination of pH (10 mg/kg) and cisplatin 0.5 mg/kg) reduced tumour mass by 75% (p=0.05).

EXAMPLE 3 Toxicity—Dehydroequol and Cisplatin

No overt signs of toxicity were noted at any of the dosage regimens used as shown in FIG. 8. Fluctuations in body mass were within ethically acceptable boundaries.

EXAMPLE 4 XIAP Expression in Human Melanoma Tumours

The differences in XIAP expression between benign and malignant melanoma tissue was assessed.

Methods

The melanoma tissue microarrays were constructed using a total of 232 primary melanomas, 15 local recurrences and 299 metastatic cores, each measuring 0.6 mm in diameter. The cohort was constructed from paraffin-embedded, formalin-fixed tissue blocks obtained from the Yale University Department of Pathology Archives. Specimens and clinical information were collected under the guidelines and approval of a Yale University Institutional Review Board. Age at diagnosis ranged from 18 to 91 years (mean age 52.4 years). The cohort included 55% males and 45% females, A pathologist reviewed slides from all of the blocks to select representative areas of invasive tumor to be cored. The cores were placed on the tissue microarray using a Tissue Microarrayer (Beecher Instruments, Silver Spring, Md.). The tissue microarrays were then cut to 0.5 μm sections and placed on glass slides using an adhesive tape-transfer system (Instrumedics, Inc., Hackensack, N.J.) with UV cross-linking. Similarly a tissue microarray was made containing cores from 540 benign nevi. The nevus array contained 31 metastatic specimens from patients that were also represented on the melanoma array. Both arrays contained identical cell lines. The overlapping metastatic specimens and cell lines were used for normalization of the scores obtained from the benign and malignant arrays.

Staining was performed for automated analysis of melanoma specimens. Slides were deparaffinized in xylene, and transferred though two changes of 100% ethanol. For antigen retrieval, the slides were boiled in a pressure cooker containing 6.5 mM sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked in a mixture of methanol and 2.5% hydrogen peroxide for thirty minutes at room temperature. To reduce non-specific background staining, slides were incubated at room temperature for 30 minutes in 0.3% bovine serum albumin/1X Trisbuffered saline. Slides were incubated at 4° C. overnight in a humidity tray with a primary mouse anti-human XIAP antibody (BD Transduction Laboratories) diluted 1:50. To create a tumor mask, slides were simultaneously incubated overnight with a primary rabbit anti-human S100 antibody diluted 1:500. Slides were rinsed three times in 1X Tris-buffered saline/0.05% Tween-20 and incubated for 1 hour at room temperature with goat anti-mouse HRP to identify the target and goat anti-rabbit IgG conjugated to Alexa 546 to identify the S100 mask. The slides were washed again as above and incubated for ten minutes with Cy5 directly conjugated to tyramide (Perkin Elmer, Boston, Mass.) at a dilution of 1:50 for primary antibody identification. The slides were rinsed again and coverslips were mounted with ProLong Gold antifade reagent, which contained 4,6-diamidine-2-phenylindole (DAPI) to identify the nuclei.

Images were acquired using automated, quantitative analysis. Briefly, areas of tumor were distinguished from stroma by creating a mask with the S100 signal tagged with Alexa 546. Coalescence of S100 at the cell surface was used to identify the membrane/cytoplasm compartment within the tumor mask, while 4,6-diamidino-2-phenylindole (DAPI) was used to identify the nuclear compartment within the tumor mask. The target marker, XIAP, was visualized with Cy5 (red). Cy5 was used because its emission peak is outside the color spectrum of tissue autoflourescence. Multiple monochromatic, high resolution (1024×1024 pixel 0.5-μm) grayscale images were obtained for each histospot, using the 10× objective of an Olympus AX-51 epifluorescence microscope (Olympus, Melville, N.Y.) with an automated microscope stage and digital image acquisition driven by custom program and macro-based interfaces with IPLabs software (Scanalytics Inc., Fairfax, Va.).

Two images (one in-focus and one out-of-focus) were taken of the compartment specific tags and the target marker. A percentage of the out-of-focus image was subtracted from the in-focus image for each pixel, representing the signal to noise ratio of the image. An algorithm described as RESA (Rapid Exponential Subtraction Algorithm) was used to subtract the out-of-focus information in a uniform fashion for the entire microarray. Subsequently, the PLACE algorithm (Pixel Locale Assignment for Compartmentalization of Expression) was used to assign each pixel in the image to a specific subcellular compartment and the signal in each location is calculated. Pixels that cannot accurately be assigned to a compartment were discarded. The data were saved and subsequently expressed as the average signal intensity per-unit of compartment area. For the nuclear and membrane/cytoplasmic compartments, the image was measured on a scale of 0-255, and expressed as target signal intensity relative to the compartment area.

The JMP5 (SAS Institute Inc., Cary, N.C.) software package was used for data analyses. Continuous AQUA scores of target expression were divided by the median score and associations with clinical and pathological parameters were completed using the Chi-Square test. Comparison of expression in malignant and benign specimens, as well as comparisons between primary and metastatic specimens was performed with unpaired t-tests.

Results

Unpaired t-tests showed that expression was significantly higher in malignant versus benign tissue cores (P<0.0001), as shown in FIG. 9. Moreover, the expression of XIAP was significantly higher in metastatic specimens than in primary specimens (P<0.0001), as shown in FIG. 9. The median AQUA scores for XIAP were 9.922 in nevi specimens. 20.56 in primary lesions and 26.98 in metastatic lesions.

Table 2 demonstrates the association between high XIAP expression and commonly used clinical and pathological variables. High XIAP was associated with advanced stage (metastatic) disease and thick lesions over 2 mm (p=0.0003 and p=0.0264, respectively). TABLE 2 Association between high XIAP expression and other prognostic clinical and pathological variables. Clincial/pathological Chi-Square Variable Value P-value Disease Stage 13.071 0.003 (metastatic vs. primary) Breslow (>2 mm) 4.927 0.0264 Clark Level (IV-V) 3.559 0.1687 Age (<40 years) 0.338 0.5613 Gender (male) 1.800 0.1798 Presence of Ulceration 2.016 0.3650

EXAMPLE 5 Sensitisation of Melanoma Cells to Carboplatin by Dehydroequol and Association with XIAP Levels

Methods

Low passage (passage 3-18) melanoma cell strains were excised from tumors of patients treated at the Yale Cancer Center, obtained form the Cell Culture facility of the Yale Skin Disease Research Core Center (YSDRCC). The melanoma cell strain YUMAC was from an in transit metastasis from a male patient, YUSAC2 from a soft tissue metastasis from a male patient, and YUGEN8 from a brain metastasis of a female patient. 501 mel, 624 mel and 888 mel were provided by Dr. Steven A. Rosenberg, National Cancer Institute, Bethesda, Md. The mm127 cell line was provided by Dr Peter Parsons of the Queensland Institute of Medical Research. Cells where grown in F12 medium with 10% F13S at 37° C. in 5% CO₂. Carboplatin was purchased from Sigma Chemical Co. (St. Louis, Mo.).

Cell viability was evaluated using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay according to the manufacturer's instructions (Promega, USA). Briefly 4×10⁴ cells per well were plated in triplicate in a 96-well microtitre plate. Cells were grown to 60% confluency, at which stage the media was replaced with reduced serum phenol-depleted OPTI-MEM (Gibco™, Invitrogen Corp, Grand Island, N.Y.), and incubated for 4 hours prior to treatment. Following treatment, 20 μL of CellTiter 96 Aqueous One Solution was added to each well and the plate was incubated at 37° C. for 2 hours. Optical densities were measured at 490 nm. Values of treated cells were compared to untreated cells and reported as percent viability.

For western blot analysis cells were plated in 35 mm² Petri dishes and grown to 60% confluency for treatment. Following treatment, cells were lysed in Radioimmunoprecipitation (RIPA) buffer containing the protease inhibitor cocktail, Complete (Roche Diagnostics, Mannheim, Germany). For phospo-AKT analysis lysis was performed in 10X cell lysis buffer (Cell Signaling, Beverly, Mass.). Protein concentrations were calculated by the BCA (Bicinchoninic Acid) assay (Pierce Biotechnology, Rockford, Ill.). Protein (20 μg) was separated in a sample buffer [2.5% SDS, 10% glycerol, 5% β-mercapto-ethanol, 0.15 M Tris (pH=6.8) and 0.01% bromophenol blue] and subjected to SDS-polyacrylamide gel electrophorisis using precast 12% polyacrylamide gels and transferred to pure nitrocellulose membranes. To inhibit non-specific binding, membranes were blocked in 5% powdered milk for 1 hour at room temperature. The membranes were washed three times in PBS with 0.5% tween (PBS-T) for 10 minutes per wash and incubated with primary antibodies diluted in PBS-T with 1% milk, in a 50 mL falcon tube on a rotator overnight at 4° C. The following primary antibodies and concentrations were used: mouse anti-XIAP (BD Transduction Laboratories) at 1:1000, mouse anti-Caspase 2 (BD Biosciences) at 1:1,000, rabbit anti-Bid (Cell Signaling, Beverly, Mass.) at 1:2500, and rabbit anti-actin (Sigma) at 1:100. Following primary incubation, membranes were washed as described above and incubated with horse anti-mouse or horse anti-rabbit peroxidase (Vector Laboratories, Burlingame, Calif.), diluted 1:10,000, for 1 hour at room temperature. Membranes were washed again in PBS-T as above and washed three times in ddH20, 10 minutes per wash. Finally, proteins were visualized using enhanced chemiluminescence.

Following drug treatment of cells, 10 μg of protein in 50 μL of ddH₂O was combined with equilibrated Caspase-Glo™ 3/7, 8, or 9 reagents (Promega). After incubation for 1 hour at room temperature, luminescence was measured using a TD 20/20 Luminometer (Turner Designs, Sunnyvale, Calif.). Blank values were subtracted and fold increase in activity was calculated based on activity measured from untreated cells. Each sample was measured in duplicate.

Results

The effect of dehydroequol on three patient-derived melanoma cell strains was evaluated. A concentration of 10 μg/ml dehydroequol was used and cell viability determined by the CellTiter 96® Aqueous One Solution Cell Proliferation Assay. As shown in FIG. 10, two of the three cell strains (MAC and YUGEN8) were sensitive to 10 μg/ml of dehydroequol, while the third cell strain (YUSAC2) was resistant. Surprisingly, YUSAC2 cells grew more in the presence of dehydroequol than in media alone.

The effect of dehydroequol on XIAP expression and function was studied in one of the two dehydroequol sensitive cell strains, YUMAC, and in the resistant strain (YUSAC2). Exposure of YUMAC cells to dehydroequol decreased the level of XIAP at 4 hours compared with pretreament levels. A further decrease in XIAP levels was seen at 8 hours post treatment and no expression was observed at 24 hours (FIG. 11 a). It was then evaluated whether changes on XIAP expression affects other component of the apoptotic cascade. As shown in FIG. 11 a, activity of caspases 3, 8 and 9 was increased at 24 hours (FIG. 11 a). With the YUSAC2 cells, the degree XIAP degradation and resultant caspase activation was much less than what was observed with YUMAC cells (FIG. 11 b).

YUMAC, YUSAC2 and YUGEN8 cells were also evaluated for sensitivity to Carboplatin. The cells were treated with increasing doses of Carboplatin, ranging from 50-200 1μg/ml for 24 hours, and cell viability was determined by the CellTiter 96® Assay. As shown in FIG. 12 a, all three cell strains were resistant to Carboplatin, with YUSAC2 demonstrating the most resistance., YUMAC and YUGEN8 were also relatively resistant to Carboplatin; over 70% of the cells were viable with the highest concentration of the drug. As with dehydroequol, YUSAC2 cells grew more in the presence of Carboplatin than in medium alone. The IC₅₀ for all three cell strains was greater than 200 μg/ml.

As demonstrated in Example 2, resistance of ovarian cancer cells to Carboplatin is associated with high XIAP levels, and that this resistance can be reversed by pretreatment with dehydroequol. In the present study the inventors assessed whether pretreatment of melanoma cells with dehydroequol reverses the baseline resistance to Carboplatin and whether this reversal is associated with XIAP degradation. Melanoma cells were pre-treated with 10 μg/ml of dehydroequol for 4 hours, dehydroequol was removed from the media and the cells were treated with increasing doses of Carboplatin (50-200 μg/ml) for an additional 24 hours. As shown in FIG. 12 b, pretreatment with dehydroequol sensitized the YUMAC and YUGEN8 cells to Carboplatin, as demonstrated by a decrease in the IC₅₀ of Carboplatin for these cell strains to <50 μg/ml and <200 μg/ml, respectively. Pretreatment with dehydroequol also decreased the resistance of YUSAC2 to Carboplatin, but to a much lesser degree with the IC₅₀ for Carboplatin remaining at >200 μg/ml.

In order to further characterize the effect of dehydroequol on melanoma cells, YUMAC cells were pretreated with a shorter exposure to dehydroequol (2 hours) followed by treatment with Carboplatin. The effect on apoptotic mediators, including procaspase-2, BID (a member of the mitochondrial pathway that is activated after caspase 2 activation) and caspase-3 was assessed. Treatment with Carboplatin alone had no effect on XIAP levels, or any other component of the apoptotic cascade. Treatment with dehydroequol for 2 hours decreased XIAP expression and induced caspase 2 activation. However, when Carboplatin was added after dehydroequol pre-treatment, XIAP degradation was observed, followed by caspase 2 and Bid activation, and a significant increase on the activity of caspase 3/7 (FIG. 13).

EXAMPLE 6 Dehydroequol-topotecan Co-administration Effectively Reduces Tumor Kinetics in an A2780 Xenograft Model of EOC

To determine the effects of topotecan and dehydroequol on epithelial ovarian cancer (EOC) cells in vivo, a mouse xenograft model was established using A2780 cells by inoculating 5 wk old male Balb/c nude mice s.c. with 1×10⁶ A780 cells bilaterally (100 μL midway between the axillary and inguinal region) along the dorsal surface. Therapy commenced 8-10 days post-inoculation and individual mouse weight was measured everyday, dehydroequol was formulated as a suspension in 1% carboxymethyl cellulose (CMC) and topotecan was formulated in PBS.

Animals were randomly assigned to 6 groups: Group 1 received 1% CMC qdx10 (control group); Group 2 received 25 mg/kg dehydroequol (p.o.) qdx10; Group 3 received 2 mg/kg topotecan (i.p.) qdx5; Group 4 received 1 mg/kg topotecan (i.p.) qdx5; Group 5 received combination dehydroequol (25 mg/kg, p.o., qdx10) and topotecan (2 mg/kg, i.p., qdx5); and Group 6 received dehydroequol (12.5. mg/kg, p.o., qdx10) plus topotecan (1 mg/kg, i.p., qdx5). Dehydroequol dosed orally at 25 mg/kg strongly retarded A2780 tumor proliferation and significantly reduced terminal tumor burden (%T/C=45.7) (FIGS. 14A-B). Similarly, topotecan also significantly reduced tumor proliferation and terminal tumor burden when dosed at both 1 and 2 mg/kg (%T/C=31.6 and 16.9 respectively). More importantly, animals that received combination dehydroequol/topotecan (25 and 2 mg/kg, respectively) had significantly reduced tumor kinetics and terminal tumor burden (%T/C=8.15) at levels below that of the corresponding monotherapy controls. Furthermore, animals that received combination dehydroequol/topotecan at doses half that of the monotherapy controls (12.5 mg/kg dehydroequol and 1 mg/kg topotecan) had significantly reduced tumor proliferation kinetics and terminal tumor burden (%T/C=−14.25) when compared to monotherapy controls (FIGS. 14A-B). These data indicate that dehydroequol and topotecan dosed in combination act synergistically in reducing overall tumor burden using the A2780 ovarian cancer tumor model. Additionally, the animals receiving the low-dose combination did not show significant weight loss (data not shown) indicating that dehydroequol-topotecan combination is able to act synergistically in preventing tumor proliferation with minimal effect on weight loss. Further, animals receiving the high-dose combination showed less myelosuppression than animals receiving topotecan monotherapy (data not shown), suggesting that dehydroequol may protect against topotecan-induced myelosuppression.

These examples highlight the utility of the isoflavonoid compounds of formula (I) in combination with chemotherapeutic agents, and the compounds of formula (II) or (IIa) and (I) as therapeutic agents for inducing sensitivity to chemoresistant cancer cells and tumours to low levels of chemotherapy and to the general down regulation of cell proliferation and the treatment, amelioration, defence against, prophylaxis and/or prevention of the therapeutic indications.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The inventions also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour. 

1. A method of increasing the sensitivity of melanoma cells or a melanoma to a chemotherapeutic agent by contacting said cells or melanoma with an isoflavonoid compound of formula (I):

in which R₁, R₂ and Z are independently hydrogen, hydroxy, OR₉, OC(O)R₁₀, OS(O)R₁₀, CHO, C(O)R₁₀, COOH, CO₂R₁₀, CONR₃R₄, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, alkylaryl, alkoxyaryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo, or R₂ is as previously defined, and R₁ and Z taken together with the carbon atoms to which they are attached form a five-membered ring selected from

or R₁ is as previously defined, and R₂ and Z taken together with the carbon atoms to which they are attached form a five-membered ring selected from

and W is R₁, A is hydrogen, hydroxy, NR₃R₄ or thio, and B is selected from

W is R₁, and A and B taken together with the carbon atoms to which they are attached form a six-membered ring selected from

W, A and B taken together with the groups to which they are associated are selected from

W and A taken together with the groups to which they are associated are selected from

and B is selected from

wherein R₃ is hydrogen, alkyl, arylalkyl, alkenyl, aryl, an amino acid, C(O)R₁₁ where R₁₁ is hydrogen, alkyl, aryl, arylalkyl or an amino acid, or CO₂R₁₂ where R₁₂ is hydrogen, alkyl, haloalkyl, aryl or arylalkyl, R₄ is hydrogen, alkyl or aryl, or R₃ and R₄ taken together with the nitrogen to which they are attached comprise pyrrolidinyl or piperidinyl, R₅ is hydrogen, C(O)R₁₁ where R₁₁ is as previously defined, or CO₂R₁₂ where R₁₂ is as previously defined, R₆ is hydrogen, hydroxy, alkyl, aryl, amino, thio, NR₃R₄, COR₁₁ where R₁₁ is as previously defined, CO₂R₁₂ where R₁₂ is as previously defined or CONR₃R₄, R₇ is hydrogen, C(O)R₁₁ where R₁₁ is as previously defined, alkyl, haloalkyl, alkenyl, aryl, arylalkyl or Si(R₁₃)₃ where each R₁₃ is independently hydrogen, alkyl or aryl, R₈ is hydrogen, hydroxy, alkoxy or alkyl, R₉ is alkyl, haloalkyl, aryl, arylalkyl, C(O)R₁₁ where R₁₁ is as previously defined, or Si(R₁₃)₃ where R₁₃ is as previously defined, R₁₀ is hydrogen, alkyl, haloalkyl, amino, aryl, arylalkyl, an amino acid, alkylamino or dialkylamino, the drawing “- - -” represents either a single bond or a double bond, T is independently hydrogen, alkyl or aryl, X is O, NR₄or S, and Y is

wherein R₁₄, R₁₅ and R₁₆ are independently hydrogen, hydroxy, OR₉, OC(O)R₁₀, OS(O)R₁₀, CHO, C(O)R₁₀, COOH, CO₂R₁₀, CONR₃R₄, alkyl, haloalkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, thio, alkylthio, amino, alkylamino, dialkylamino, nitro or halo, or any two of R₁₄, R₁₅ and R₁₆ are fused together to form a cyclic alkyl, aromatic or heteroaromatic structure, and pharmaceutically acceptable salts thereof
 2. The method of claim 1 wherein the melanoma cells or melanoma display resistance to the chemotherapeutic agent.
 3. The method of claim 2, wherein the sensitivity of the melanoma cells or melanoma to the chemotherapeutic agent is restored as a result of the treatment.
 4. The method of claim 1, wherein the compound of formula (I) is administered to a subject in need of such treatment
 5. The method of claim 1 wherein the chemotherapeutic agent is selected from carboplatin, cisplatin, paclitaxel, docatexel, gemcitabine, and topotecan.
 6. The method of claim 5 wherein the chemotherapeutic agent is carboplatin.
 7. The method of claim 1 wherein the compound of formula (I) is dehydroequol.
 8. A method for the treatment or prevention of melanoma in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I) as defined in claim 1 and a chemotherapeutic agent.
 9. The method of claim 8 wherein the compound of formula (I) is administered prior to the chemotherapeutic agent.
 10. The method of claim 8 wherein the compound of formula (I) and the chemotherapeutic agent are administered simultaneously.
 11. The method of claim 8 wherein the therapy follows observed resistance in the melanoma cells or melanoma to a chemotherapeutic agent.
 12. The method of claim 8 wherein the chemotherapeutic agent is selected from carboplatin, cisplatin, paclitaxel, docataxel, gemcitabine and topotecan.
 13. The method of claim 12 wherein the chemotherapeutic agent is carboplatin.
 14. The method of claim 8 wherein the compound of formula (I) is dehydroequol.
 15. Combination therapy for the treatment or prevention of melanoma comprising administering to a subject a therapeutically effective amount of a compound of formula (I) as defined in claim 1 and a chemotherapeutic agent.
 16. A pharmaceutical composition for the treatment or prevention of melanoma, the composition comprising at least one isoflavonoid compound of formula (I) and at least one chemotherapeutic agent.
 17. The composition of claim 16 wherein the chemotherapeutic agent is selected from: carboplatin, cisplatin, paclitaxel, docatexel, gemcitabine and topotecan.
 18. The composition of claim 16 wherein the compound of formula (I) is dehydroequol. 